核酸酶NucB高效表达工程菌株的构建及功能测定
Construction and functional evaluation of an engineered bacte-rial strain for high-efficiency expression of NucB nuclease
李江勇 1陈熙明 2刘光琇 2张威 2陈拓 3袁亚玲 1李善家1
作者信息
- 1. 兰州理工大学 生命科学与工程学院,甘肃 兰州 730050
- 2. 中国科学院西北生态环境资源研究院 沙漠与沙漠化重点实验室,甘肃 兰州 730000;甘肃省极端环境微生物资源与工程重点实验室,甘肃 兰州 730000
- 3. 中国科学院西北生态环境资源研究院 冰冻圈科学国家重点实验室,甘肃 兰州 730000;甘肃省极端环境微生物资源与工程重点实验室,甘肃 兰州 730000
- 折叠
摘要
目的 构建可以高效稳定表达核酸酶NucB的工程菌株.方法 以大肠杆菌DH5α(DE3)为材料,采用基因编辑的方法将其中的必需基因lexA敲除,重新设计一个带有lexA基因的质粒进行基因回补,从而得到工程菌株DH5α(DE3)ΔlexA,再将带有目的基因nucB的质粒转入基因改造后的菌株中发酵培养生产核酸酶NucB并测定功能.结果 经过基因改造后的大肠杆菌DH5α(DE3)ΔlexA与未经基因改造的大肠杆菌对比显示:改造后的菌株质粒非常稳定,蛋白表达量高且发酵过程中可以不用添加抗生素来生产核酸酶NucB.结论 以基因编辑的方法成功构建一株无抗生素、高效稳定表达核酸酶NucB的工程菌株DH5α(DE3)ΔlexA.
Abstract
Objective To objective of this study was to construct an engineered bacterial strain capable of effi-ciently and stably expressing NucB nuclease.Methods The DH5α(DE3)strain of Escherichia coli was used as the host organism.Gene knockout was performed using genetic editing techniques to disrupt the essential gene lexA within the bacterial genome.Subsequently,a modified plasmid containing the lexA gene was designed and introduced to complement the knockout,resulting in the engineered strain DH5α(DE3)ΔlexA.A plasmid carrying the target gene nucB was then transformed into the modified strain for fermentation cultivation and production of NucB nuclease,followed by functional characterization.Results A comparative analysis between the genetically modified strain DH5α(DE3)ΔlexA and the wild-type Escherichia coli revealed that the modi-fied strain exhibited highly stable plasmids,significantly increased protein expression levels,and the ability to produce NucB nuclease during fermentation without the need for antibiotic supplementation.Conclusion In summary,we successfully constructed an engineered strain by using genetic editing methods,DH5α(DE3)ΔlexA,which enables antibiotic-free,efficient,and stable expression of NucB nuclease.
关键词
基因敲除/无抗生素/高效稳定/核酸酶NucBKey words
gene knockout/antibiotic-free/efficient and stable/NucB nuclease引用本文复制引用
基金项目
甘肃省科技重大专项(22ZD6WA035)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据